Agrobacterium-mediated gene transduction is a method for transforming plants through the use of a function of Agrobacterium. A soil bacterium Agrobacterium tumefaciens functions in such a manner that the T-DNA which forms a part of its Ti (tumor-inducing) plasmid involved in the pathogenicity of the Agrobacterium, is integrated into the genome of a plant when it infects the plant. Agrobacterium-mediated plant transformation is a method of introducing a desired gene into the genome of a plant through the use of the function of Agrobacterium described above by constructing a transforming plasmid in which the T-DNA region of the Ti plasmid is replaced by the gene desired to be introduced into the plant genome, and using Agrobacterium prepared to carry the transforming plasmid in place of the Ti plasmid.
Agrobacterium-mediated plant transformation was originally developed mainly as a method for transforming dicotyledons because Agrobacterium were thought to infect only dicotyledons but not monocotyledons. Various attempts for Agrobacterium-mediated gene transduction into monocotyledons were also made, and super-binary vectors having a part of the virulent genes of super-virulent Agrobacterium strains were developed and reported to be useful for stably transforming even monocotyledons such as rice and maize with relatively high efficiency (Hiei, et al., 1994; Ishida, et al., 1996; Japanese Patent No. 2,649,287; Japanese Patent No. 3,329,819). Successful examples of Agrobacterium-mediated transformation of monocotyledons such as wheat, barley and sorghum were also reported, and Agrobacterium-mediated transformation also became to be widely applied to monocotyledons.
Agrobacterium-mediated transformation generally has many advantageous features such as high efficiency, low copy number transgenes, transferability of such a specific region as T-DNA without being fragmented, and short-term culture for obtaining transformants resulting in little somaclonal variations. Thus, it is widely used as the most useful means for transforming many plant species irrespective of whether they are dicotyledonous or monocotyledonous.
Agrobacterium-mediated transformation is equally performed in all plants by contacting a material tissue with an Agrobacterium suspension, coculturing them and then selecting a transformed cell to produce a transformed plant, though the materials and the compositions of the culture media vary with plant species. Typically, the plant tissue used as a material is infected with Agrobacterium without any special treatment except for optional sterilization (Rogers et al. 1988, Visser 1991, McCormick 1991, Lindsey et al. 1991).
Agrobacterium-mediated transformation has been reported for many plant species, but has the disadvantage that the transformation efficiency widely varies with plant species, genotypes and material tissues (Potrykus et al. 1998). It is important to develop a technology enabling transformed plants to be stably obtained with high efficiency throughout the year because many transformed plants must be produced when a variety containing a practical gene is to be cultured. Moreover, transformation methods independent from plant species and genotypes would be very useful for efficiently culturing practical varieties. Additionally, transformation methods independent from plant tissues used as materials would be required for efficient transformation.
As described above, it is important to develop a method capable of improving gene transduction efficiency or transforming plant species or genotypes involving difficulty in gene transduction, and many reports have already existed. However, many of them focus on the modification of the compositions of culture media, marker genes or promoters or the investigation of materials. Some reports discussed methods for treating plant tissues before infection with Agrobacterium to adapt them for gene transduction, but all of them contemplated improving infection efficiency by injuring tissues via incision (Chan et al. 1993), particle gun (Tingay et al. 1997), sonication (Trick and Finer 1997, Amoah et al. 2001), enzyme treatment (Weber et al. 2003), etc.
Hiei et al. found that plant tissues are transformed with higher efficiency by Agrobacterium-mediated gene transduction after centrifugation as compared with untreated tissues (WO 02/12520, Japanese Patent Laid-Open Publication No. 2000-342256). Although the details of the mechanism via which transformation efficiency is improved by centrifugation are unknown, it is thought that cells turned into a physiological state suitable for gene transduction by centrifugation unlike the methods of physically injuring tissues as described above. It was also found that tissues having undergone heat treatment or both of centrifugation and heat treatment are transformed with higher efficiency as compared with untreated tissues (Japanese Patent Laid-Open Publication No. 2000-342255, Japanese Patent Laid-Open Publication No. 2000-342253).
Teasdale et al. filed a patent application for a method of transformation comprising immersing a plant tissue in a medium including an infective transformation vector and reducing and/or raising the pressure (WO 99/48335). Teasdale et al. describe that the over pressure is designed to promote infiltration of the transformation vector into the plant tissue. However, they describe only an experimental example of a treatment under negative pressure, but no example of treatment under positive pressure. Thus, no data is shown to support that pressurization is effective for improving gene transduction efficiency.
Pullman and Peter filed a patent application for a method of improving the frequency of embryogenic callus formation by culturing a plant tissue under pressure (U.S. Pat. No. 6,492,174). They describe that culturing at 1.5 atm gave the highest frequency of callus formation in the experiment at pressures of 1-2.5 atm for the culture period of 8 weeks. All the other experiments were performed at a very low positive pressure such as 1.5 atm. There is no description about the effect of pressurization on gene transduction.    Patent Publication No. 1: International Publication No. WO 02/12520.    Patent Publication No. 2: International Publication No. WO 99/48355.    Patent Publication No. 3: U.S. Pat. No. 6,492,174.    Patent Publication No. 4: Japanese Patent No. 2,649,287.    Patent Publication No. 5: Japanese Patent No. 3,329,819.    Patent Publication No. 6: Japanese Patent Laid-open Publication No. 2000-342256.    Patent Publication No. 7: Japanese Patent Laid-open Publication No. 2000-342255.    Patent Publication No. 8: Japanese Patent Laid-open Publication No. 2000-342253.    Patent Publication No. 9: International Publication No. WO 95/06722.    Patent Publication No. 10: International Publication No. WO 03/027290.    Non-Patent Publication No. 1: Amoah, B. K., Wu, H., Sparks, C. and Jones, H. D. (2001) Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. Journal of Experimental Botany 52: 1135-1142.    Non-Patent Publication No. 2: Chan, M-T., Cheng, H-H., Ho, S-L., Tong, W-F. and Yu, S-M. (1993) Agrobacterium-mediated production of transgenic rice plants expressing a chimeric a-amylase promoter/β-glucuronidase gene. Plant Molecular Biology, 22, 491-506.    Non-Patent Publication No. 3: Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T. (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6, 271-282.    Non-Patent Publication No. 4: Hoekema, A., Hirsch, P. R., Hooykaas, P. J. J. and Schilperoort, R. A. (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature, 303, 179-180.    Non-Patent Publication No. 5: Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Komari, T. and Kumashiro, T. (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotech. 14: 745-750. Non-Patent Publication No. 6: Komari, T. (1990) Genetic characterization of a double-flowered tobacco plant obtained in a transformation experiment. Theor. Appl. Genet. 80: 167-171.    Non-Patent Publication No. 7: Komari, T. and Kubo, T. (1999) Methods of Genetic Transformation: Agrobacterium tumefaciens. In Vasil, I. K. (ed.) Molecular improvement of cereal crops. Kluwer Academic Publishers, Dordrecht, pp. 43-82.    Non-Patent Publication No. 8: Lindsey, K., Gallois, P. and Eady, C. (1991) Regeneration and transformation of sugarbeet by Agrobacterium tumefaciens. Plant Tissue Culture Manual B7: 1-13. Kluwer Academic Publishers.    Non-Patent Publication No. 9: McCormick, S. (1991) Transformation of tomato with Agrobacterium tumefaciens. Plant Tissue Culture Manual B6: 1-9. Kluwer Academic Publishers.    Non-Patent Publication No. 10: Potrykus, I., Bilang, R., Futterer, J., Sautter, C. and Schrott, M. (1998) Agricultural Biotechnology, NY: Mercel Dekker Inc. pp. 119-159.    Non-Patent Publication No. 11: Rogers, S. G., Horsch, R. B. and Fraley, R. T. (1988) Gene transfer in plants: Production of transformed plants using Ti plasmid vectors. Method for Plant Molecular Biology, CA: Academic Press Inc. pp. 423-436.    Non-Patent Publication No. 12: Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang, M., Thornton, S. and Brettell, R. (1997) Agrobacterium tumefaciens-mediated barley transformation. The Plant Journal 11: 1369-1376.    Non-Patent Publication No. 13: Trick, H. N. and Finer, J. J. (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation. Transgenic Research 6: 329-336.    Non-Patent Publication No. 14: Visser, R. G. F. (1991) Regeneration and transformation of potato by Agrobacterium tumefaciens. Plant Tissue Culture Manual B5: 1-9. Kluwer Academic Publishers.    Non-Patent Publication No. 15: Weber, S., Friedt, W., Landes, N., Molinier, J., Himber, C., Rousselin, P., Hahne, G. and Horn, R. (2003) Improved Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.): assessment of macerating enzymes and sonication. Plant Cell Reports 21: 475-482.